Single-cell analysis reveals a subpopulation of adipose progenitor cells that impairs glucose homeostasis

Adipose progenitor cells (APCs) are heterogeneous stromal cells and help to maintain metabolic homeostasis. However, the influence of obesity on human APC heterogeneity and the role of APC subpopulations on regulating glucose homeostasis remain unknown. Here, we find that APCs in human visceral adipose tissue contain four subsets. The composition and functionality of APCs are altered in patients with type 2 diabetes (T2D). CD9+CD55low APCs are the subset which is significantly increased in T2D patients. Transplantation of these cells from T2D patients into adipose tissue causes glycemic disturbance. Mechanistically, CD9+CD55low APCs promote T2D development through producing bioactive proteins to form a detrimental niche, leading to upregulation of adipocyte lipolysis. Depletion of pathogenic APCs by inducing intracellular diphtheria toxin A expression or using a hunter-killer peptide improves obesity-related glycemic disturbance. Collectively, our data provide deeper insights in human APC functionality and highlights APCs as a potential therapeutic target to combat T2D. All mice utilized in this study are male.

The authors have identified an adipose progenitor cell (APC) sub population in human visceral adipose tissue that participates in Type 2 Diabetes (T2D) development.By comparing the abundance of APC subpopulations in lean, obese and obese T2D, it appeared that the CD9 APC subset was higher in obese and T2D patients compared to lean donors.Then, different approaches strongly suggest that the CD9+CD55low subset is associated with the development of T2D, and an elegant series of experiments functionally demonstrated that this APC subpopulation plays a crucial role in driving T2D progression in diet-induced obese mice.Finally, a detailed analysis of secretomes revealed that this pathological APC subpopulation displays a chemotactic capacity and regulates adipose lipolysis, both capacities tested in vitro and in vivo.The manuscript is clearly written and reports a very significant work in the field.Data support the conclusions.However, some claims should be modulated (see below).The methodology is sound.
2) A high abundance of PDGFRα+CD9high APCs in adipose tissues of obese patients has been previously reported.This subpopulation correlates with fibrosis level of the tissue and with T2D (Marcellin et al. 2017; Ref 14 in the submitted manuscript).Therefore, the sentence "APC CD9 cells appear to be a novel subset of APC in human visceral adipose tissue" (p5, line 20) should be modulated.
3) According to the FACS-based selection of APCs illustrated in Fig. 2b, the sub population having a potential fibrotic behaviour could be CD9-CD55-giving rise to ICAM1+ cells.How the PDGFRα+CD9high could be mapped in the CD9+CD55high sub populations should be more discussed.
4) Images presented in Fig. 3a and g are of a weak quality and should be improved to be more convincing.
5) FACS analyses reports the maintenance of the human APCs several weeks after transplantation in immunocompetent mice, likely thanks to the adoptive cell transfer approach used by the authors.The approach used has to be described in details. 6) Authors performed experiments in obese mice to deplete all the APC subpopulations and to investigate their role in obesity-related metabolic disorders.They conclude that APCs depletion is sufficient to achieve metabolic benefits.This conclusion should be more discussed and the authors should propose how to integrate in this model data showing that APCs are critical to generate new adipocytes in response to pathological conditions (Virtue, S.; Vidal-Puig, A. Adipose Tissue Expandability, Lipotoxicity and the Metabolic Syndrome-An Allostatic Perspective.Biochim.Biophys.Acta 2010, 1801, 338-349.s).Previously, Dacquinag et al. (Cell Death and Differentiation, 2015) performed a similar experiment using a peptide D-WAT, similar than this used in the submitted manuscript, and showed that APC depletion prevents dyslipidemia.The authors proposed that the metabolic benefit of the APC depletion was due to beiging of white adipose tissue.These data should also be incorporated in the discussion.

Reviewer #2 (Remarks to the Author):
The article " scRNA-seq reveals a subpopulation of adipose progenitor cell that impairs glucose homeostasis" by H.Wang et al. identifies CD9+CD55low adipocyte progenitor cells (APC) as a cell subpopulation increased in human obesity-related T2D and suggest that these cells have a direct detrimental impact on adipose tissue and systemic metabolism.The regulatory APC populations have been proposed by several previous studies and this one is an additional report that such cell populations might exist.Both human APCs and multiple murine models are employed and the study presents interesting findings with potential therapeutic impact.However, there are few pitfalls that need to be addressed before strong conclusions regarding the role of CD9+CD55low cell population can be drawn.The major problem are the lack of correct controls in the cell transplantation experiments and the lack of specificity towards a particular APC population of cells (in this case CD9+CD55low) in the APC depletion experiments.
These are specific comments: Transplantation experiments.
-Data shown in figure 3 are obtained only from 3-5 animals and unfortunately doesn't have a suitable control.The injection of human cells is a large stress factor and could have caused inflammation and immune response that subsequently could have affected WAT and systemic insulin sensitivity.Cells that are not expected to contribute to hyperglycemia should have been used as a control (for example CD55high) and effects caused by CD9+CD55low transfer should have been compared to the effects obtained with the injection of other APC population.PBS is not a suitable control and there is a large possibility that the results are misleading.o For the experiments shown in 3a, were the percentages of remaining APCs quantified?-Transplantation data presented in figure 5 also lack reliable control.The control should have been CD9+CD55low cell population from lean individuals.Mice that didn't receive any transplantation cannot serve a control for transplanted ones due to multiple side effects of such intervention.If the authors state, that CD9+CD55low population is changing phenotype in obesity/T2D, the direct comparison of this cell population from lean and obese/T2D patients would be needed.
Flow cytometry characterization of APCs -Please add CD34 expression in your progenitor violin plots 1d as a general marker for human adipocyte progenitors.
-This reviewer would like to see FMO controls for CD31, PDGFRA, CD55, CD9, ICAM and CD142 in the same picture as 2a.The definition of positive cells is not quite obvious and flow cytometry is not well-described in the method section.
Depletion/treatment experiments.Major problem with the depletion experiments using D-WAT and tamoxifen treatments are non-specific depletion of all APC populations therefore although these data proves the importance of APCs in general they don't address a specific role of CD9+CD55low cell populations -Figure 6. Do the mice gain weight on HFD between moths 3 and 4 after they are treated with tamoxifen or vehicle (fig 6)?The result that depletion of APCs improves metabolic phenotype goes against multiple previous data where studies suggested that differentiation of new adipocytes is metabolically beneficial compared to the increased size of mature adipocytes.Could it be that tamoxifen injection and APC depletion has non-specific effects that are associated with metabolic phenotype.Could the authors show the data of weight, food intake and fat cell size in the PD mice injected with tamoxifen on HFD.In addition, how can the authors exclude that tamoxifen alone has a metabolic effect when mice are on HFD.There was no effect on chow, but metabolic conditions are changing substantially in obesity.-Figure 7.In the treatments of DIO and db/db mice with D-WAT, which APC population was depleted?Please present the data of depletion for different APC populations?What was food intake rate, what was the weight of these mice after the treatment.Bioinformatic analysis -Extended figure 2. Could you please include references for clusters shown in ext fig.2c,d.The references are missing in both the text and the figure legend therefore it's difficult to understand that the authors mean… Do the authors map their clusters to cells obtained from visceral or subcutaneous clusters.-CD9 cells do not correspond to any of previously identified clusters.Could the authors comment on this fact?What is the difference from the multiple other studies and the ones shown in extended figure 2? What about purity of the sample?-CD9-/CD55-/ICAM1-population was decreased in obese and obese T2D patients.What are these cells?Do they express CD34?PDRFRA?Can they differentiate or if this decrease only relative as CD9+ cell population is increased?Other: -Could you please change colors in figure 1c to visualize contribution of each patient to the clusters, -In general, figure legends needs to be expanded to .In many cases, it is not clear what is shown, and n numbers are lacking.Please add n to each panel where relevant.
-The lightness of the microscopy pictures should be increased.It is impossible to see the staining (neither on the screen, nor in the printed version).
-The method section does not provide all the necessary details to reproduce the data shown.

Reviewer #3 (Remarks to the Author):
Wang, et al. utilize single-cell RNA sequencing (scRNA-seq) to study adipose tissue composition of type 2 diabetes (T2D) patients.The key findings are: 1. T2D patients have altered adipose progenitor cell (APC) composition.2. The CD9+ APCs population is a distinct APC subpopulation and contributes to T2D development.3. Depletion of CD9+ APCs in mice improves obesity-related glycemic disturbances.
The role of adipose progenitors in the development of T2D is poorly understood.Wang, et al. seek to address this critical question through the application of single-cell RNA-sequencing technology and in vivo APC transfer assays.The study demonstrates significant dedication to the recruitment, processing, and analysis of clinically relevant donor samples.As presented, however, the findings presented in this manuscript should be better supported with a more comprehensive interpretation of existing single-cell data, a critical review of underlying assumptions, and improvement in the experimental design.
Major concerns: • In Fig. 1, the authors characterize the stromal vascular fractions derived from human visceral adipose tissues.It is important to recognize that the captured transcriptomic profiles represent a snapshot of the dynamic tissue.Throughout the manuscript, APCs_CD9 is depicted as a stable subpopulation with a distinct role.However, the slingshot trajectory analysis (Fig. 1g) positions the APCs_CD9 cluster in a transition state, situated between the APCs_CD55 progenitors and the further developed APCs_ICAM1 (committed preadipocytes) and APCs_CD142 (adipogenesis-regulatory cells).The APCs_CD9 cells have intermediate levels of marker gene expressions (Fig. 1d) and enrichment (Fig. 1h, Extended data fig.2c,d) in between those of early progenitors and developed cells.Altogether, the profile of APCs_CD9 cells resembles a dynamic intermediate developmental cell state and not a static pathogenic cell population.
• In the subsequent figures, the authors designed experiments with the assumption that CD9+CD55low is a distinct cell type.In Fig. 4 and Fig. 5, the authors focus solely on comparing CD9+CD55low cells derived from lean and obese T2D donors.It is important to consider the possibility that the distinctions observed between lean and obese donors may not be exclusive to CD9+ cells but could potentially extend to all APCs.
• An appropriate control group is missing in the APCs transfer experiments presented in Fig. 3, Fig. 5g-n, and Extended data Fig. 5f,g.The use of PBS as a control does not appropriately control for the impact of allogenic cell injection.Does the injection of CD9-CD55high cells also induce lipolysis?
• The authors claim that the majority of APCs in the adipose tissue of obese mice are CD9+ cells, and consequently, the metabolic effects induced by the ablation of all APCs presented in Fig. 6 and Fig. 7 are attributed to the removal of CD9+ APCs.The assumption is based on the flow cytometry profile derived from a diet-induced obesity mouse model, which suggests that CD9+ APCs make up ~75% of the total APCs in the obese mouse model.However, this attribution is valid only if the ablation of CD9-APCs has no discernable physiological impact.
• The authors have integrated the scRNA-seq results from this study with previously published APCs datasets (page 5, line 17-18, Fig. 1h, Extended data fig.2c,d).However, the authors did not indicate which reference studies were used.
• On page 6, in line 9, the authors have misinterpreted the PCA plot.They assert that CD9+CD55low APCs exhibit the most distinct separation from the rest of the dataset.However, it is important to note that the first principal component, responsible for 40% of the dataset's variance, distinguishes the CD55+ population from the other populations.The second principal component, which accounts for 26% of the variance, is the one that distinguishes the CD9+ population.Therefore, it is the CD55+ population that exhibits the most obvious separation from the others.
• In Fig. 4c, the authors have elected to only highlight proteins enriched in the control/non-T2D group.However, given that the primary focus is on examining the secretome of APCs derived from T2D patients, it would be more informative to highlight specific proteins enriched in the T2D group to be highlighted.Additionally, the protein names are presented as two clusters, raising the question of whether the heatmap is aligned correctly with the protein annotations.
• Different marker genes for the APCs_CD9 cells are employed in different.Genes analyzed by qPCR, as depicted in Extended Data Fig. 3b, show limited overlap with the APCs_CD9 marker genes presented in Extended Data Fig. 2a, or the CD9+CD55low APCs marker genes displayed in Fig. 2d.
• The authors should offer a rationale for selecting the APCs_CD55 cluster as the root state of the Slingshot pseudo-time analysis.
• On page 9, line 19, it should read "little" instead of "litter." • The authors should clarify in the figure legends the meaning of the blue data points in the volcano plot presented in Fig. 5a.We thank the reviewer for raising this point.
To determine whether CD55 + APC is related to CD26 + APC, the signature genes of

Response:
In accord with the reviewer's suggestion, we removed this sentence in the revised manuscript.

Response:
We appreciate your comment.
As illustrated in Supplementary Fig. 2b, genes related to fibrosis were not enriched in APCs_ ICAM1.GO analysis further indicated that genes associated with the extracellular structure organization pathway were not enriched in the APCs_ ICAM1 (Supplementary Fig. 2c).
PDGFRα + CD9 high APCs reported by Marcellin et al. exhibited two dominant features: high expression of CD9 and pro-fibrosis capacity.In our study, as depicted in Supplementary Fig. 8d, expression of genes related to fibrosis was highly upregulated in CD9 + CD55 low APCs from T2D patients compared to that from lean subjects.Thus, CD9 + CD55 low APCs identified in our study corresponds to PDGFRα + CD9 high APCs reported by Marcellin et al.
We have included this description in the Discussion section (Page 23, lines 7-9).4. Images presented in Fig. 3a and g are of a weak quality and should be improved to be more convincing.

Response:
Thank you very much.
We have replaced the micrographs with better quality of the figures (Fig. 3a, Fig. 3g, Fig. 6k, Fig. 7h, and Fig. 7s) in the revised manuscript.

FACS analyses reports the maintenance of the human APCs several weeks after transplantation in immunocompetent mice, likely thanks to the adoptive cell
transfer approach used by the authors.The approach used has to be described in details.

Response:
In response to this suggestion, we have added the details of adoptive cell transfer to the Method section (Page 25, lines 9-21; Page 33, lines 17-21; Page 34, lines 1-9).

Response:
We thank the reviewer for raising these valuable comments.Although we could not rule out the possibility that APC depletion may interfere with adipogenesis, the influence may be minor.The reasons are: (1) Previous studies using different lineage tracing mouse models investigated the timing of APC activation and adipogenesis in response to HFD, showing that new adipocytes form in eWAT between five and eight weeks of HFD feeding 1,2 .In this study, mice were fed a HFD for more than eight weeks before APC were depleted.So, by the time of APC depletion, adipogenesis may not be affected.( 2) Another study has reported that CD9 + APCs are the predominant subset in the eWAT of obese mice and can hardly differentiate into mature adipocyte 3 .Consistent with previous studies, depleted APCs in this study were mainly CD9 + APCs.Nevertheless, whether APC depletion impedes adipocyte generation is an important issue to investigate.Accordingly, we have added the following text in the Discussion PBS is not a suitable control and there is a large possibility that the results are misleading.

Response:
Thank you for your valuable suggestion.Your constructive suggestion was very helpful for us to improve the quality of this work.
In accord with the reviewer's suggestion, we included CD9 -APCs from T2D patients as a control group.The results showed that mice received CD9 + CD55 low APCs transplantation had increased fasting glucose and impaired glucose tolerance compared to that received CD9 -APCs transplantation (Fig. 3d-f, m-o).

For the experiments shown in 3a, were the percentages of remaining APCs quantified?
Response: Thank you for raising this question.
The frequencies of remaining APC subpopulations were detected by flow cytometry (Supplementary Fig. 4a-c).After D-WAT treatment, frequencies of both CD9 + APCs and CD9 -APCs in the eWAT of C57BL/6 mice were decreased.These data have been included in the revised manuscript (Page 9, lines 11-13).5

Response:
Thanks for your constructive comments.
We used CD9 + CD55 low APCs from lean control subjects as the control.Compared to the mice transplanted with CD9 + CD55 low APCs from lean control subjects, mice transplanted with CD9 + CD55 low APCs from obese T2D patients showed higher levels of serum FFA and glycerol (Fig. 5k,l).Accordingly, we have revised the text in the Result section (Page 13, line 20-22; Page 14, lines 1-6).

Response:
Thanks for your advice.Expression of CD34 has been included in Fig. 1d in the revised manuscript.

This reviewer would like to see FMO controls for CD31, PDGFRA, CD55, CD9,
ICAM and CD142 in the same picture as 2a.The definition of positive cells is not quite obvious and flow cytometry is not well-described in the method section.

Response:
Thanks for your positive comments.
All FMO controls have been added in the revised manuscript (Supplementary Fig. 3b).Meanwhile, more details about flow cytometry have been added in the Method section (Page 31, lines 15-22; Page 32, lines 11-17).

Depletion/treatment experiments. Major problem with the depletion experiments using D-WAT and tamoxifen treatments are non-specific depletion of
all APC populations therefore although these data proves the importance of APCs in general they don't address a specific role of CD9+CD55low cell populations.

Response:
We appreciate your valuable comments to improve our experiments.Currently, it is technically hard to deplete one specific APC subpopulation.Therefore, we reconstituted CD9 -APCs in APC-depleted mice to exclude the influence of CD9 -APC depletion on glycemic metabolism: 1.In obese PD mice, approximately 3.5×10 4 CD9 -APCs were depleted after tamoxifen treatment.So, as shown in Fig. 6a, CD9 -APCs from obese PD mice were transplanted into APC-depleted mice.In agreement with our findings in Fig. 3 that CD9 - APCs did not impair glycemic homeostasis, APC depletion significantly improved glycemic disturbance in obese PD mice, whereas CD9 -APC recovery did not alter these metabolic benefits.

Response:
Thanks for your comments.
As shown in supplementary Fig. 6j, after tamoxifen or vehicle treatment, body weight was not statistically different among groups of obese PD mice.
Although we could not rule out the possibility that APC depletion may interfere with adipogenesis, the influence may be minor.The reasons are: (1) Previous studies using different lineage tracing mouse models investigated the timing of APC activation and adipogenesis in response to HFD, showing that new adipocytes form in eWAT between five and eight weeks of HFD feeding 1,2 .In this study, mice were fed a HFD for more than eight weeks before APC were depleted.So, by the time of APC depletion, adipogenesis may not be affected.( 2) Another study has reported that CD9 + APCs are the predominant subset in the eWAT of obese mice and can hardly differentiate into mature adipocyte 3 .Consistent with previous studies, depleted APCs in this study were mainly CD9 + APCs.In addition, to further determine whether tamoxifen injection per se could alter glycemic metabolism, PD mice and Pdgfra-CreERT2 mice fed a HFD for 3 months were treated with tamoxifen or vehicle (Reply Fig. 2a).As shown in the following figure, four weeks after tamoxifen or vehicle treatment in PD mice, glycemic disturbance was notably improved in tamoxifen treated mice compared to mice injecting vehicle (Reply Fig. 2b,c).However, tamoxifen treatment had no effect in HFD-fed Pdgfra-CreERT2 mice, as compared to PD mice injecting vehicle.These data indicate that tamoxifen injection per se could not impact glycemic metabolism.8. Could the authors show the data of weight, food intake and fat cell size in the PD mice injected with tamoxifen on HFD.In addition, how can the authors exclude that tamoxifen alone has a metabolic effect when mice are on HFD.There was no effect on chow, but metabolic conditions are changing substantially in obesity.

Response:
Thanks for your valuable comments.
The data of body weight, food intake and adipocyte size in APC-depleted PD mice have been added in the Supplementary Fig. 6i-m.Food intake and body weight were not statistically different among groups of obese PD mice (Supplementary Fig. 6i-k).HE staining showed that the eWAT of APC-depleted mice contained a higher proportion of large adipocytes compared to the vehicle-treated mice (Supplementary Fig. 6l,m).These important data have been incorporated into the Results section in the revised manuscript (Page 15, lines 11-12, 19-21).
In addition, as we replied to the above comment, glycemic disturbance was not improved after tamoxifen injection in obese Pdgfra-CreERT2 mice, indicating that tamoxifen administration per se could not alter glycemic metabolism.

Response:
We appreciate these helpful comments.
To address the Reviewer's question, we analyzed the effect of D-WAT on APC frequency, food intake and body weight.As illustrated in Fig. 7 and Supplementary Fig.

Response:
Thanks for your valuable comment.
The relevant references have been cited in the revised manuscript.The adipose tissue depot used for analysis was added in the figure legends (Fig. 1g, Supplementary Fig. 2e,f).Accordingly, these references were incorporated in the main text (Page 6, line 11).

CD9 cells do not correspond to any of previously identified clusters. Could the authors comment on this fact? What is the difference from the multiple other studies and the ones shown in extended figure 2? What about purity of the sample?
Response: We appreciate this insightful question raised by the reviewer.
To better address this important issue, we performed additional analysis.In this study, to map our APC clusters with previous findings, we selected three elegant studies which have been incorporated into the main text (Page 6, line 11).Among these studies, findings from two studies (Merrick et al. and Schwalie et al.) contribute to the consensus of APC heterogeneity in mouse, while Emont et al. conducted single-cell sequencing on human visceral and subcutaneous adipose tissues.To better visualize our APC data compared to the published literature, we integrate our single-cell dataset with those reported by above studies and performed unsupervised cell clustering.We found that four APC subpopulations identified in our study could be finely distinguished in the integrated dataset.Moreover, Icam1+ committed progenitor and Dpp4+ multipotential progenitor identified by individual studies were generally preserved after integration.In agreement, as shown in Fig. 1g and Supplementary Fig. 2e,f, APCs_CD55 cells resembled multipotent progenitors; APCs_ICAM1 cells expressed a gene signature similar to that of committed preadipocytes; APCs_CD142 cells tended to be adipogenesis-regulatory cells, whereas APCs_CD9 did not correspond well with a particular subpopulation.
Based on these findings, we conclude that APCs_CD9 do not correspond to any of previously identified clusters.The main text has been revised (Page 6, lines 10-14).
12. CD9-/CD55-/ICAM1-population was decreased in obese and obese T2D patients.What are these cells?Do they express CD34?PDRFRA?Can they differentiate or if this decrease only relative as CD9+ cell population is increased?

Response:
Thanks for your valuable comments.
CD34 was expressed in CD9 -CD55 -ICAM1 -APC subpopulations, as validated by both single-cell analysis (Reply Fig. 3a) and flow cytometry (Reply Fig. 3b).Furthermore, in response to standard differentiation factors in vitro, these cells can differentiate into adipocyte (Reply Fig. 3c).
In addition, as shown in Fig. 2e, we analyzed the relative frequency of each APC subpopulation in total APC.So, due to the increased frequency of CD9 + CD55 low APCs, the frequency of CD9 -CD55 -ICAM1 -APCs was decreased in obese and T2D patients.
Promoted by your comments, the expression level of CD34 has been added in Fig. 1d in the main text.
Reply Figure 3

Response:
As suggested, we changed colors in Fig. 1c and added a new figure depicting the contribution of each donor (Supplementary Fig. 2a).Thank you.

Response:
We thank the reviewer for the opportunity to clarify and improve our figure legends.
As suggested, we have added more details in the figure legends.

Response:
Thanks for your comment.
Additional experimental details have been included in the Methods section to strengthen the understanding and reproducibility of this study.

Reviewer # 1 :
Major concerns: 1. Fig.1h: CD55+ APCs are reported as multipotent progenitors.Is this population similar to the CD26+ APC subpopulation previously reported (Merrick et al. 2019)?Response: CD55 + APC were compared to that of three APC subsets defined by Merrick et al.As shown in Fig. 1g, the signature genes of APCs_CD55 subpopulation were enriched in CD26 + (encoded by DPP4) APC subpopulation, which was previously identified as multipotent progenitor cell by Merrick et al.We have added the relevant reference in the revised manuscript (Page 6, line 11).Moreover, we compared the expression of signature genes of CD26 + APC subpopulation reported by Merrick et al. to the four APC subsets discovered in our study.As shown in reply Figure 1, the highest expression of signature genes of CD26 + APC was detected in our APCs_CD55 subpopulation, but not in other three subpopulations.These data support that APCs_CD55 subpopulation strongly resembled previously identified CD26 + APC subpopulation.Reply Figure 1: Comparison of the data reported by Merrick et al. with our dataset.A heatmap of the signature genes of the DPP4 + APC expressed in the four APC subsets defined in our study.

2 .
A high abundance of PDGFRα+CD9high APCs in adipose tissues of obese patients has been previously reported.This subpopulation correlates with fibrosis level of the tissue and with T2D (Marcellin et al. 2017; Ref 14 in the submitted manuscript).Therefore, the sentence "APC CD9 cells appear to be a novel subset of APC in human visceral adipose tissue" (p5, line 20) should be modulated.
Fig.2b, the sub population having a potential fibrotic behavior could be CD9-CD55-giving rise to ICAM1+ cells.How the PDGFRα+CD9high could be mapped in the CD9+CD55high sub populations should be more discussed.
to deplete all the APC subpopulations and to investigate their role in obesity-related metabolic disorders.They conclude that APCs depletion is sufficient to achieve metabolic benefits.This conclusion should be more discussed and the authors should propose how to integrate in this model data showing that APCs are critical to generate new adipocytes in response to pathological conditions (Virtue, S.; Vidal-Puig, A. Adipose Tissue Expandability, Lipotoxicity and the Metabolic Syndrome-An Allostatic Perspective.Biochim.Biophys.Acta 2010, 1801, 338-349.s).Previously, Dacquinag et al. (Cell Death and Differentiation, 2015) performed a similar experiment using a peptide D-WAT, similar than this used in the submitted manuscript, and showed that APC depletion prevents dyslipidemia.The authors proposed that the metabolic benefit of the APC depletion was due to beiging of white adipose tissue.These data should also be incorporated in the discussion.

1 .
section (Page 20, lines 3-5):"In addition, it is worth noting that APCs are critical to generate new adipocytes during the progression of obesity, whether APC depletion impedes adipocyte generation needs further investigation."References:1.Wang QA, Tao C, Gupta RK, Scherer PE.Tracking adipogenesis during white adipose tissue development, expansion and regeneration.Nature medicine 19, 1338-1344 (2013).2. Jeffery, E., Church, C.D., Holtrup, B., Colman, L. & Rodeheffer, M.S. Rapid depotspecific activation of adipocyte precursor cells at the onset of obesity.Nature cell biology 17, 376-385 (2015).3. Marcelin, G., et al.A PDGFRalpha-Mediated Switch toward CD9(high) Adipocyte Progenitors Controls Obesity-Induced Adipose Tissue Fibrosis.Cell metabolism 25, 673-685 (2017).We appreciate the second issue raised by the reviewer.Study from Dacquinag et al. indicates that APC depletion could induce beige adipocyte differentiation.The experimental strategy performed in their study was different from that of ours.They depleted mouse APCs through s.c.injection of D-WAT peptide before HFD feeding was given, whereas APCs were depleted through in situ injection in eWAT of obese mice in our study.Since functionality of APC changes during obesity progression, depleting APCs at different stage of obesity may cause different consequences.As suggested, we have added the following sentences in the Discussion section (Page 20, lines 5-8): "Moreover, another study reported that APC depletion before the development of obesity could induce beige adipocyte differentiation, indicating that depleting APCs at different stage of obesity may cause diverse consequences."Data shown in figure 3 are obtained only from 3-5 animals and unfortunately doesn't have a suitable control.The injection of human cells is a large stress factor and could have caused inflammation and immune response that subsequently could have affected WAT and systemic insulin sensitivity.Cells that are not expected to contribute to hyperglycemia should have been used as a control (for example CD55high) and effects caused by CD9+CD55low transfer should have been compared to the effects obtained with the injection of other APC population.
also lack reliable control.The control should have been CD9+CD55low cell population from lean individuals.Mice that didn't receive any transplantation cannot serve a control for transplanted ones due to multiple side effects of such intervention.If the authors state, that CD9+CD55low population is changing phenotype in obesity/T2D, the direct comparison of this cell population from lean and obese/T2D patients would be needed.

7. Figure 6 .
Do the mice gain weight on HFD between moths 3 and 4 after they are treated with tamoxifen or vehicle (fig 6)?The result that depletion of APCs improves metabolic phenotype goes against multiple previous data where studies suggested that differentiation of new adipocytes is metabolically beneficial compared to the increased size of mature adipocytes.Could it be that tamoxifen injection and APC depletion has non-specific effects that are associated with metabolic phenotype.

Reply Figure 2 :
Glycemic disturbance was not improved by tamoxifen treatment in obese Pdgfra-CreERT2 mice.a, PD mice and Pdgfra-CreERT2 mice were fed a HFD for 3 months and treated with tamoxifen (TMX).One month after tamoxifen treatment, GTT assay (b) and ITT assay (c) were conducted (n=5 per group).*, P<0.05; **, P<0.01 (One-way ANOVA followed by Tukey's multiple comparison test).

9. Figure 7 .
In the treatments of DIO and db/db mice with D-WAT, which APC population was depleted?Please present the data of depletion for different APC populations?What was food intake rate, what was the weight of these mice after the treatment.

7
, after D-WAT treatment, both CD9 + APCs and CD9 -APCs were reduced in DIO and db/db mice, whereas no significant differences were observed on food intake and body weight.These important data have been incorporated in the revised manuscript (Page 16, lines 21-22; Page 17, lines 13-14).Bioinformatic analysis 10. Extended figure 2. Could you please include references for clusters shown in ext fig.2c,d.The references are missing in both the text and the figure legend therefore it's difficult to understand that the authors mean.Do the authors map their clusters to cells obtained from visceral or subcutaneous clusters.
: a, Violin plot showing the expression level of CD34 in four APC subpopulations; b, Detection of CD34 expression in CD9 -CD55 -ICAM1 -APCs by flow cytometry; c, CD9 -CD55 -ICAM1 -APCs were sorted by flow cytometry and cultured in vitro.Twelve days after standard differentiation factors treatment, adipogenic differentiation was analyzed by BODIPY staining.Other: 13.Could you please change colors in figure 1c to visualize contribution of each patient to the clusters.
legends needs to be expanded.In many cases, it is not clear what is shown, and n numbers are lacking.Please add n to each panel where relevant.
Fig.7s) with high quality images in the revised manuscript.